WO2021147837A1 - 抗lag3单克隆抗体及其制备方法和应用 - Google Patents
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- A—HUMAN NECESSITIES
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- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the invention relates to the field of biomedicine, in particular to an anti-LAG3 monoclonal antibody and its preparation method and application.
- LAG3 (Lymphocyte-activation gene 3), also called CD223, was discovered in 1990. It belongs to the immunoglobulin superfamily and is expressed on activated T cells, NK cells, B cells and dendritic cells.
- MHC major histocompatibility complex
- MHC II major histocompatibility complex
- the affinity of LAG3 and MHC II is even stronger than that of CD4 and MHC II.
- the ligands of LAG3 also include LSECtin and FGL1.
- the biological function of LAG3 is similar to CTLA-4 and PD-1, and it plays a role in negatively regulating the cell proliferation and activation of T cells.
- LAG3 also plays a role in the suppressive function of Treg cells.
- T cells can be reactivated, thereby enhancing the killing effect on tumors.
- inhibiting LAG3 can also reduce the function of Treg cells to suppress the immune response. Therefore, using LAG3 as a target for cancer immunotherapy, the use of monoclonal antibodies to bind LAG3 can block the binding of LAG3 to its ligand and prevent the generation of T cell suppression signals. Thereby promoting the activation and proliferation of T cells, as well as the expression of cytokines, up-regulating the monitoring activity of the immune system against tumor cells, enhancing the specific anti-tumor immune response, and achieving the purpose of treating tumors.
- anti-LAG3 antibody BMS986016 researched and developed by Bristol-Myers Squibb has achieved significant results in the treatment of advanced melanoma and is currently undergoing phase III clinical trials.
- anti-LAG3 antibodies are used in combination with other antibody drugs including anti-PD-1 antibodies, and related clinical trials are also being carried out.
- the technical problem to be solved by the present invention is to provide a new anti-LAG3 monoclonal antibody, thereby completing the present invention.
- the first objective of the present invention is to provide a new anti-LAG3 monoclonal antibody.
- the second object of the present invention is to provide a nucleotide molecule encoding the anti-LAG3 monoclonal antibody.
- the third object of the present invention is to provide an expression vector containing the nucleotide molecule.
- the fourth object of the present invention is to provide a host cell containing the expression vector.
- the fifth object of the present invention is to provide a method for preparing the anti-LAG3 monoclonal antibody.
- the sixth object of the present invention is to provide a composition containing the anti-LAG3 monoclonal antibody.
- the seventh objective of the present invention is to provide the application of the anti-LAG3 monoclonal antibody in the preparation of medicines.
- the first aspect of the present invention provides an anti-LAG3 monoclonal antibody, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising SEQ ID NO: 2, 6
- the heavy chain variable region shown in 10 has HCDR1, HCDR2, and HCDR3 regions with the same CDR sequence, and the light chain variable region includes the same as those shown in SEQ ID NO: 4, 8 or 12.
- the anti-LAG3 monoclonal antibody includes:
- the HCDR1 has the amino acid sequence shown in SEQ ID No: 18, the HCDR2 has the amino acid sequence shown in SEQ ID No: 19, and the HCDR3 has the amino acid sequence shown in SEQ ID No: 19, The amino acid sequence shown in SEQ ID No: 20 or 21;
- the light chain complementarity determining regions LCDR1, LCDR2, LCDR3, the LCDR1 has the amino acid sequence shown in SEQ ID No: 22 or 23, the LCDR2 has the amino acid sequence shown in SEQ ID No: 24, and the LCDR3 It has the amino acid sequence shown in SEQ ID No: 25.
- the anti-LAG3 monoclonal antibody includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 2, 6, or 10. Or a sequence having at least 85% homology with the aforementioned sequence, such as a derivative sequence with 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% homology;
- the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 4, 8 or 12, or a sequence with at least 85% homology with the above sequence, such as 85%, 90%, 92%, 94%, 95 %, 96%, 97%, 98%, or 99% homology of derived sequences.
- the anti-LAG3 monoclonal antibody includes a heavy chain and a light chain, and the heavy chain is composed of an amino acid sequence as shown in SEQ ID NO: 2, 6 or 10 or at least 85% identical to the above sequence.
- Source sequences such as heavy chain variable regions of 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% homology, and such as SEQ ID NO:
- the heavy chain constant region shown in 14 is composed;
- the light chain is composed of an amino acid sequence shown in SEQ ID NO: 4, 8 or 12, or a sequence having at least 85% homology with the above sequence, such as 85%,
- amino acid sequence of the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 2
- amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.
- amino acid sequence of the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.
- amino acid sequence of the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 10
- amino acid sequence of the light chain variable region is shown in SEQ ID NO: 12.
- the anti-LAG3 monoclonal antibody may be the full-length sequence of the antibody, or may be a fragment of an anti-LAG3 antibody, and the fragment is Fab, Fab', F(ab')2, Fv, scFv, or the like.
- the anti-LAG3 antibody is an IgG1, IgG2 or IgG4 type antibody.
- the present invention further provides a derivative of the anti-LAG3 antibody, the derivative is a fragment of the LAG3 antibody, an antibody/antibody fragment-factor fusion protein, an antibody/antibody fragment-chemical conjugate; the fragment of the anti-LAG3 antibody is Fab, Fab', F(ab')2, Fv or scFv, etc.
- the monoclonal antibodies of the present invention can be prepared by conventional techniques in the art, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
- monoclonal antibodies are prepared from wild-type or transgenic mice by hybridoma technology.
- the second aspect of the present invention provides an isolated nucleotide molecule encoding the anti-LAG3 monoclonal antibody as described above.
- the nucleotide sequence encoding the heavy chain variable region of the anti-LAG3 monoclonal antibody is as shown in SEQ ID NO: 3, 7 or 11, which encodes the light chain variable region of the anti-LAG3 monoclonal antibody
- the nucleotide sequence of is shown in SEQ ID NO: 5, 9 or 13.
- nucleotide sequence encoding the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the light chain variable region of the anti-LAG3 monoclonal antibody As shown in SEQ ID NO: 5.
- nucleotide sequence encoding the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 7, and the nucleotide sequence encoding the light chain variable region of the anti-LAG3 monoclonal antibody As shown in SEQ ID NO: 9.
- nucleotide sequence encoding the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 11, and the nucleotide sequence encoding the light chain variable region of the anti-LAG3 monoclonal antibody As shown in SEQ ID NO: 13.
- the preparation method of the nucleotide molecule is a conventional preparation method in the art, and preferably includes the following preparation method: obtaining the nucleotide molecule encoding the monoclonal antibody by gene cloning technology, such as PCR method, or by artificial full sequence
- the synthetic method obtains the nucleotide molecule encoding the above-mentioned monoclonal antibody.
- nucleotide sequence encoding the amino acid sequence of the monoclonal antibody can be replaced, deleted, changed, inserted or added as appropriate to provide a polynucleotide homologue or conservative variant sequence thereof.
- the homologue of the polynucleotide of the present invention or its conservative variant sequence can be prepared by replacing, deleting or adding one or more bases encoding the monoclonal antibody gene within the scope of maintaining antibody activity.
- the third aspect of the present invention provides an expression vector containing the nucleotide molecule as described above.
- the expression vector is a conventional expression vector in the art, which means that it contains appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other appropriate Sequence expression vector.
- the expression vector may be a virus or a plasmid, such as an appropriate phage or phagemid.
- a virus or a plasmid such as an appropriate phage or phagemid.
- Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.
- the expression vector in the present invention refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors well known in the art.
- the expression vector is selected from one or more of pHLX101, pEE14.4, pCHO 1.0 or pcDNA3.1.
- the expression vector is pcDNA3.1.
- the fourth aspect of the present invention provides a host cell containing the expression vector as described above.
- the host cell of the present invention is a variety of conventional host cells in the field, as long as it can make the above-mentioned recombinant expression vector stably replicate itself and the nucleotides carried can be effectively expressed.
- the host cell in the present invention can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
- the host cell is: COS, CHO (Chinese Hamster Ovary), HeLa cell line, bone marrow cell line such as SP2/0 cell line, NS0, sf9, sf21, DH5 ⁇ , BL21(DE3) or E .coli TG1, YB2/0 cell line, etc. and one or more of transformed B-cells or hybridoma cells.
- the host cell is E. coli TG1, BL21 cell (expressing single-chain antibody or Fab antibody) or CHO-K1 cell (expressing full-length IgG antibody).
- the expression vector is transformed into a host cell to obtain the preferred recombinant expression transformant of the present invention.
- the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electrotransformation method.
- the fifth aspect of the present invention provides a method for preparing the anti-LAG3 monoclonal antibody as described above, including the following steps:
- the isolation and purification method of the anti-LAG3 monoclonal antibody is a conventional method in the art.
- specific operation method please refer to the corresponding cell culture technology manual and the monoclonal antibody isolation and purification technology manual.
- the host cells used in the present invention are all existing technologies and can be directly obtained through commercial channels.
- the culture medium used in the culture is also various conventional culture media. Those skilled in the art can select the appropriate medium based on experience.
- the host cell is cultured under conditions where it grows. After the host cell has grown to a suitable cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
- the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art.
- Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
- the present invention screens and obtains the gene sequence of the target antibody from a monoclonal cultured cell line, and is used to construct a eukaryotic expression vector. After expression, the activity of the antibody can be reconstructed to obtain an anti-LAG3 monoclonal antibody.
- the sixth aspect of the present invention provides a composition comprising the anti-LAG3 monoclonal antibody as described above and a pharmaceutically acceptable carrier.
- the monoclonal antibody of the present invention can be used by formulating a pharmaceutical composition in any manner known in the art.
- This composition takes the monoclonal antibody as the active ingredient, plus one or more pharmaceutically acceptable carriers, diluents, fillers, binding agents and other excipients, depending on the mode of administration and the design The dosage form.
- the therapeutically inert inorganic or organic carriers known to those skilled in the art include (but are not limited to) lactose, corn starch or its derivatives, talc, vegetable oils, waxes, fats, polyanthocyanidin compounds such as polyethylene glycol, water , Sucrose, ethanol, glycerin, etc., various preservatives, lubricants, dispersing agents, flavoring agents.
- Moisturizers antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like can also be added. These substances are used as needed to help the stability of the formulation or help increase the activity or its biological effectiveness or In the case of oral administration, it produces acceptable taste or smell.
- the inhibitor may be used in the form of its original compound itself, or optionally in the form of its pharmaceutically acceptable salt.
- the cloned antibody can be administered alone, or in various combinations, and in combination with other therapeutic agents.
- the composition thus formulated can be used to administer the inhibitor in any appropriate manner known to those skilled in the art as required.
- the anti-LAG3 monoclonal antibody provided by the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition to achieve a more stable therapeutic effect.
- These preparations can ensure the amino acid core sequence of the anti-LAG3 monoclonal antibody of the present invention Conformational integrity, while also protecting the protein's multifunctional groups to prevent its degradation (including but not limited to aggregation, deamination or oxidation).
- it can be stored at 2°C-8°C for at least one year, and for freeze-dried preparations, it can be kept stable at 30°C for at least six months.
- the anti-LAG3 monoclonal antibody preparation can be a suspension, water injection, freeze-dried and other preparations commonly used in the pharmaceutical field, preferably a water injection or freeze-dried preparation,
- the pharmaceutically acceptable carrier preferably includes, but is not limited to: one of surfactants, solution stabilizers, isotonic regulators and buffers or Its combination.
- the surfactants preferably include but are not limited to: non-ionic surfactants such as polyoxyethylene sorbitol fatty acid ester (Tween 20 or 80); Poloxamer (such as Poloxamer 188); Triton; sodium dodecyl sulfate (SDS) ); sodium lauryl sulfate; tetradecyl, linoleyl or octadecyl sarcosine; Pluronics; MONAQUATTM, etc., the amount added should minimize the tendency of anti-LAG3 monoclonal antibody to granulate.
- non-ionic surfactants such as polyoxyethylene sorbitol fatty acid ester (Tween 20 or 80); Poloxamer (such as Poloxamer 188); Triton; sodium dodecyl sulfate (SDS) ); sodium lauryl sulfate; tetradecyl, linoleyl or octa
- the solution stabilizer preferably includes but is not limited to one or a combination of the following: sugars, for example, reducing sugars and non-reducing sugars; amino acids, for example, monosodium glutamate or histidine; alcohols, for example : Triols, higher sugar alcohols, propylene glycol, polyethylene glycol, etc.
- the amount of solution stabilizer added should enable the final formulation to maintain a stable state within a time period considered by those skilled in the art to be stable.
- the isotonicity adjusting agent preferably includes, but is not limited to, one of sodium chloride, mannitol, or a combination thereof.
- the buffer preferably includes but is not limited to one of Tris, histidine buffer, phosphate buffer, or a combination thereof.
- the seventh aspect of the present invention provides the application of the above-mentioned anti-LAG3 monoclonal antibody or its composition in the preparation of medicines.
- the application is for preparing LAG3 molecular blocker drugs. More preferably, the use of the preparation of LAG3 molecular blocker drugs is specifically the preparation of tumor treatment or tumor diagnosis drugs.
- the drug of the present invention is preferably an anti-tumor, treatment of autoimmune diseases, treatment of infectious diseases and/or anti-transplant rejection, more preferably anti-tumor drugs, treatment of autoimmune diseases, and more preferably For anti-tumor drugs.
- the anti-LAG3 monoclonal antibody of the present invention can be used alone or in combination with other anti-tumor drugs.
- the other anti-tumor drugs are conventional anti-tumor drugs in the field, including antibody drugs or small molecule anti-tumor drugs.
- the antibody drugs are conventional antibody drugs in the art, and preferably include anti-PD-1 monoclonal antibodies.
- the small molecule anti-tumor drugs are conventional drugs in the field, including paclitaxel, 5-Fu pyrimidine and the like.
- the tumors targeted by the anti-tumor drugs preferably include but are not limited to: melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, One or more of esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma, pancreatic cancer, and/or head and neck tumors.
- the anti-tumor drugs referred to in the present invention refer to drugs capable of inhibiting and/or treating tumors, which may include delays in the development of tumor-related symptoms and/or reduction in the severity of these symptoms, and further include alleviation of symptoms associated with existing tumors. And to prevent the appearance of other symptoms, including reducing or preventing tumor metastasis and so on.
- the dosage of administration varies with the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. You can refer to the animal As a result of the experiment and various circumstances, the total dose should not exceed a certain range. Generally, the dose for intravenous injection is 1-1800 mg/day.
- the anti-LAG3 monoclonal antibody of the present invention has good biological activity, high expression in mammalian cells, and has obvious affinity for LAG3 and the ability to inhibit ligand-receptor binding.
- the monoclonal antibody can be used alone or in combination with other anti-tumor drugs in tumor immunotherapy, diagnosis and screening, and can be effectively used in the preparation of drugs for the treatment of tumors, infectious diseases, autoimmune diseases, and anti-immune rejection. .
- Figure 1 shows the experimental results of the binding ability of anti-LAG3 antibodies to LAG3 on the cell surface
- Figure 2 shows the experimental results of anti-LAG3 antibody blocking the binding of LAG3 to its ligand MHC II
- Figure 3 shows the experimental results of the binding ability of anti-LAG3 chimeric antibodies to LAG3
- Figure 4 shows the experimental results of the ability of anti-LAG3 chimeric antibody to bind to LAG3 on the cell surface
- Figure 5 shows the experimental results of the binding ability of anti-LAG3 humanized antibodies to LAG3;
- Figure 6 shows the experimental results of the binding ability of anti-LAG3 humanized antibodies to LAG3 on the cell surface
- Figure 7 shows the experimental results of surface plasmon resonance of the affinity of anti-LAG3 humanized antibody to LAG3;
- Figure 8 shows the experimental results of anti-LAG3 humanized antibodies blocking the binding of LAG3 to its ligand MHC II;
- Figure 9 shows the experimental results of anti-LAG3 humanized antibodies blocking the binding of LAG3 to its ligand LSECtin
- Figure 10 shows the experimental results of anti-LAG3 humanized antibodies blocking the binding of LAG3 to its ligand FGL1;
- Figure 11 shows the results of an ELISA experiment of species cross-reaction of anti-LAG3 humanized antibody
- Figure 12 shows the results of a flow cytometry experiment of species cross-reaction of anti-LAG3 humanized antibody
- Figure 13 shows the experimental results of surface plasmon resonance of the species cross-reaction of anti-LAG3 humanized antibody
- Figure 14 shows the experimental results of the activation of T cells by anti-LAG3 humanized antibodies
- Figure 15 shows the experimental results of anti-LAG3 humanized antibodies inhibiting tumor growth in the hLAG3 KI mouse model
- Figure 16 shows that the combination of h6H11B10#40 and HLX10 activates the release of IL-2;
- Figure 17 shows that the combination of h6H11B10#40 and HLX10 activates the release of IFN- ⁇ ;
- Figure 18 shows the growth curve of the tumor volume of each group of mice in the MC38 model
- Figure 19 shows the growth curve of the tumor volume of each group of mice in the A20 model
- the following examples further illustrate the present invention, but the present invention is not limited to the scope of the described examples.
- the experimental methods without specific conditions are selected in accordance with conventional methods and conditions, or in accordance with the product specification.
- the room temperature described in the embodiment is a conventional room temperature in the art, and is generally 10-30°C. Unless otherwise specified, all reagents and raw materials used are commercially available.
- variable region of an antibody refers to the variable region (VL) of the antibody light chain or the variable region (VH) of the antibody heavy chain, alone or in combination.
- VL variable region
- VH variable region
- the variable regions of the heavy chain and the light chain each consist of 4 framework regions (FR) connected by 3 complementarity determining regions (CDR) (also called hypervariable regions).
- FR framework regions
- CDR complementarity determining regions
- the CDRs in each chain are held together tightly by FRs and together with the CDRs from the other chain contribute to the formation of the antigen binding site of the antibody.
- There are at least two techniques for determining CDRs (1) Methods based on cross-species sequence variability (ie, Kabat et al.
- CDR may refer to a CDR determined by either method or a combination of the two methods.
- antibody framework or "FR region” refers to a part of a variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.
- CDR complementarity determining region
- CDR-H1, CDR-H2, CDR-H3 three CDRs
- CDR-L1, CDR-L2, CDR-H3 three CDRs
- Any one of various well-known schemes can be used to determine the amino acid sequence boundaries of CDRs, including the "Kabat” numbering rule (see Kabat et al.
- the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3);
- the CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3).
- the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residue numbers in VL are 26-32 (LCDR1), 50- 52 (LCDR2) and 91-96 (LCDR3).
- CDR is defined by amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24-35 in human VL.
- 34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) constitute.
- the CDR amino acid residue numbers in VH are roughly 26-35 (HCDR1), 51-57 (HCDR2) and 93-102 (HCDR3)
- the CDR amino acid residue numbers in VL are roughly 27-32 (LCDR1) ), 50-52 (LCDR2) and 89-97 (LCDR3).
- the CDR region of an antibody can be determined using the program IMGT/DomainGap Align.
- LAG3 refers to the lymphocyte activation gene-3 protein, an immune checkpoint receptor or T cell co-suppressor, also known as CD223.
- the amino acid sequence of the full length LAG3 is provided in GenBank under the accession number NP_002277.4.
- the term "" includes variants, isoforms, homologs, orthologs and paralogues.
- the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. Conventional technology. These technologies have been fully explained in the existing literature.
- Positive control antibody BMS986016 The amino acid sequence is recorded in Bristol-Myers Squibb's patent application WO 2015/042246 Al.
- the present invention clones the nucleotide sequence encoding the amino acid sequence into the pURO vector by restriction endonucleases, transfects the plasmid into CHO-S cells, and obtains high-expressing cell strains by screening for carbenicillin resistance. Expand the culture of the cell line, collect the medium supernatant, and obtain the control antibody through the preparation process of protein A affinity chromatography.
- Negative control HLX10 (h1G4): its sequence is quoted from CN109923126A.
- Positive control ab40465 Abcam company, item number ab40465.
- Isotype-Human IgG4 (hIgG4): Sino-American Crown Corporation, Cat: AB180018
- Recombinant mouse LAG3-Fc fusion protein purchased from Yiqiao Shenzhou, Cat:53069-M02H.
- Recombinant cynomolgus monkey LAG3-Fc fusion protein purchased from Yiqiao Shenzhou, Cat:90841-C08H
- Anti-mouse PD1 antibody RMP1-14 BioXCell, Cat: 717918O1
- LAG3-His amino acid sequence shown in SEQ ID No: 1
- CHO-S cells expressing the full length of LAG3 were used to co-immunize mice, and spleen cells and myeloma cells were fused to obtain After hybridoma cells, positive clones were then screened by ELISA.
- the nucleic acid sequence encoding the 23-450 amino acids of the extracellular region of LAG3 was cloned into the pEE14.4 vector by restriction endonuclease, wherein the C-terminus of the LAG3 coding region had a tag encoding 6xHis, and then the plasmid It was electroporated into CHO-S cells, and a single clone with high expression of LAG3-his fusion protein was screened by the GS (Glutamine Synthetase) screening system. The clone was expanded and cultured, and the culture supernatant was collected and passed through nickel. The method of column affinity chromatography prepares the target protein.
- the protein and CHO-S cells expressing the full length of LAG3 were co-immunized with mice.
- the LAG3-His recombinant protein was first coated in a 96-well half-well plate (working volume 30 ⁇ l) at 2 ⁇ g/ml, and coated overnight at 4°C. Wash 3 times with PBS (PBST) containing 0.05% Tween 20 to remove excess LAG3-His. Block with 5% skim milk in PBS for 1 hour at room temperature.
- the flow cytometry method was further used to determine the positive hybridoma clones that were screened by ELISA and express antibodies.
- the normally grown CHO-S cells expressing the full-length human LAG3 gene were washed twice with PBS (2% FBS), and the cell concentration was adjusted to 5 ⁇ 10 5 /100 ⁇ l.
- the cells were washed twice with PBS (2% FBS).
- the cells were suspended in 200 ⁇ l PBS and analyzed by flow cytometry. The results are shown in Figure 1. Most of the positive clones screened by ELISA can bind to LAG3 on the cell surface.
- Flow cytometry was used to test the ability of the antibodies expressed by the positive clones of hybridomas screened by ELISA to block the binding of MHC II on the cell surface to LAG3-Fc (Acro). Wash Daudi cells twice with PBS (2% FBS) and adjust the cell concentration to 3 ⁇ 10 5 /100 ⁇ l. The fusion tumor supernatant was mixed with LAG3-Fc at a concentration of 2 ⁇ g/ml, then mixed with Daudi cells, and incubated at 4°C for 30min. Centrifuge the cells at 500 ⁇ g for 5 min. At the same time, no antibody was used as a negative control, and the anti-LAG3 antibody BMS986016 was used as a positive control. Wash twice with PBS (2% FBS).
- amino acid sequence of the heavy chain variable region of 6H11B10 is shown in SEQ ID No: 2, and the nucleotide sequence is shown in SEQ ID No: 3; the amino acid sequence of the light chain variable region is shown in SEQ ID No: 4.
- the nucleotide sequence is shown in SEQ ID No:5.
- the amplification of the variable regions of the mouse light chain and heavy chain was carried out according to the method introduced by Anke Krebber et al. (Journal of Immunological Methods 201.1997.35-55).
- the general method includes preparing total RNA from the monoclonal hybridoma cell line 6H11B10, and preparing single-stranded cDNA using a reverse transcription kit (TaKaRa).
- TaKaRa reverse transcription kit
- the heavy chain variable region SEQ ID No: 3
- light chain variable region of the antibody are amplified by PCR. Region (SEQ ID No: 5).
- the light chain variable region and heavy chain variable region genes of 6H11B10 were fused with the heavy chain IgG4 constant region gene (SEQ ID No: 15) and the light chain constant region gene (SEQ ID No: 17) respectively. , Thereby obtaining the 6H11B10 chimeric antibody heavy chain gene and light chain gene.
- the chimeric antibody genes of 6H11B10 were respectively transfected into CHO-S cells and expressed to obtain the chimeric antibody protein c6H11B10.
- ELISA technique was used to detect the affinity of the chimeric antibody to LAG3.
- the LAG3-Fc recombinant protein was coated into an enzyme-labeled plate (working volume 30 ⁇ l) at 2 ⁇ g/ml, and allowed to stand overnight at 4°C. Wash 3 times with PBS (PBST) containing 0.05% Tween 20. Block with 5% skim milk in PBS for 1 hour at room temperature. Wash with PBST 3 times, then add c6H11B10 with gradient dilution, and let stand at room temperature for 1 hour.
- PBST PBS
- BMS986016 was used as a positive control. Wash with PBST 3 times, add 30 ⁇ l of horseradish peroxidase labeled goat anti-human IgG kappa light chain secondary antibody (Millipore company) diluted 1:4000, and let stand at room temperature for 1 hour. Wash 6 times with PBST, add TMB for color development, and stop the reaction with 2M H 2 SO 4 , and read at 450 nm with a microplate reader. The results are shown in Figure 3, the anti-LAG3 chimeric antibody c6H11B10 has a strong affinity for LAG3, and at low concentrations, the affinity of c6H11B10 is higher than that of the control antibody BMS986016.
- Flow cytometry was used to determine the affinity of the chimeric antibody protein c6H11B10 to LAG3 on the cell surface. Take the Jurkat cells transfected and express the full-length human LAG3 gene, wash the cells twice with PBS (2% FBS), and adjust the cell concentration to 2 ⁇ 10 5 /100 ⁇ l. Add serially diluted chimeric antibody c6H11B10 and murine antibody m6H11B10, and incubate at 4°C for 30 min. Centrifuge the cells at 500 ⁇ g for 5 min. At the same time, BMS986016 was used as a positive control. Wash twice with PBS (2% FBS).
- chimeric antibodies and BMS986016 For chimeric antibodies and BMS986016, add 1:300 diluted goat anti-human IgG Fab-FITC (Thermo Fisher), and for mouse antibodies add 1:100 diluted goat anti-mouse IgG-FITC (Kangwei Century), Incubate for 30 min at 4°C. The cells were washed twice with PBS (2% FBS). The cells were suspended in 200 ⁇ l PBS and analyzed by flow cytometry. The results are shown in Figure 4, the chimeric antibody c6H11B10 can bind strongly to LAG3 on the cell surface, and the affinity of c6H11B10 is better than that of the control antibody BMS986016.
- the human antibody light chain variable region germline gene IGKV2-28*01 and the heavy chain variable region germline gene IGHV3-11*06 were used to convert the framework of the murine antibody 6H11B10 Region replacement, retaining the original complementarity determining region (CDR), thereby obtaining the anti-6H11B10 humanized antibody h6H11B10.
- the amino acid sequence of the heavy chain variable region of h6H11B10 is shown in SEQ ID No: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 8.
- the gene sequence of the heavy chain variable region of h6H11B10 is shown in SEQ ID No: 7, and the gene sequence of the light chain variable region is shown in SEQ ID No: 9, respectively. : 15) and the constant region gene of the light chain (SEQ ID No: 17) to obtain the heavy chain gene and the light chain gene of the anti-LAG3 humanized antibody h6H11B10.
- the three CDRs of the h6H11B10 heavy chain are: HCDR1 (SEQ ID No: 18), HCDR2 (SEQ ID No: 19) and HCDR3 (SEQ ID No: 20); the three CDRs of the light chain are: LCDR1 (SEQ ID No: 22), LCDR2 (SEQ ID No: 24) and LCDR3 (SEQ ID No: 25).
- the humanized anti-LAG3 antibody h6H11B10 uses phage display technology for affinity maturation, designs primers with point mutations, and uses PCR to mutate the light chain CDRs and heavy chain CDRs of h6H11B10 to obtain a mutation library.
- the phage display vector Transform into E. coli TG1 or SS320 cells to generate a phage library.
- streptavidin-coupled magnetic beads M-280 Thermo Fisher
- biotin-labeled LAG3 protein the phage library was screened in two rounds. One clone with the highest affinity, h6H11B10#40, was screened by ELISA.
- the amino acid sequence of the heavy chain variable region of h6H11B10#40 is shown in SEQ ID No: 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 12.
- the heavy chain variable region gene of h6H11B10#40 is shown in SEQ ID No: 11, and the light chain variable region gene is shown in SEQ ID No: 13, and the heavy chain IgG4 constant region gene (SEQ ID No: 15 ) And the constant region gene of the light chain (SEQ ID No: 17) to obtain the heavy chain gene and light chain gene of the anti-LAG3 humanized antibody h6H11B10#40.
- the three CDRs of the heavy chain of h6H11B10#40 are: HCDR1 (SEQ ID No: 18), HCDR2 (SEQ ID No: 19) and HCDR3 (SEQ ID No: 21); the three CDRs of the light chain are: LCDR1 ( SEQ ID No: 23), LCDR2 (SEQ ID No: 24) and LCDR3 (SEQ ID No: 25).
- the h6H11B10 and h6H11B10#40 anti-LAG3 humanized antibody genes were respectively transfected into CHO-S cells and expressed to obtain h6H11B10 and h6H11B10#40 antibody proteins.
- ELISA technology was used to detect the affinity of humanized antibody to LAG3.
- the LAG3-Fc recombinant protein was coated into an enzyme-labeled plate (working volume 30 ⁇ l) at 2 ⁇ g/ml, and allowed to stand overnight at 4°C. Wash 3 times with PBS (PBST) containing 0.05% Tween 20. Block with 5% skim milk in PBS for 1 hour at room temperature.
- the cells were washed twice with PBS (2% FBS). The cells were suspended in 200 ⁇ l PBS and analyzed by flow cytometry. The results are shown in Figure 6, the anti-LAG3 humanized antibody has a strong affinity for LAG3 on the cell membrane, and both h6H11B10#40 and h6H11B10 are better than the positive control BMS986016.
- SPR Surface plasmon resonance
- Example 6 The ability of anti-LAG3 humanized antibody to block the binding of LAG3 to its ligand
- Flow cytometry was used to measure the ability of anti-LAG3 humanized antibodies to block the binding of MHC II on the surface of Raji cells to LAG3-Fc. Wash Raji cells twice with PBS (2% FBS) and adjust the cell concentration to 2 ⁇ 10 5 /100 ⁇ l. After the humanized anti-LAG3 antibody h6H11B10#40 is diluted 1:3, it is diluted with 2 ⁇ g/ml LAG3- After mixing the Fc, place it at room temperature for 30 minutes, while using hIgG4 protein as a negative control and anti-LAG3 antibody BMS986016 as a positive control. Then mix with Raji cells and incubate at 4°C for 60 min. Centrifuge the cells at 500 ⁇ g for 5 min.
- the anti-LAG3 humanized antibody h6H11B10#40 has the ability to block the binding of MHC II on the cell surface to LAG3-Fc, similar to BMS986016.
- ELISA technology was used to detect the ability of humanized antibodies to block the binding of LAG3-Fc to its ligand LSECtin.
- the LAG3-Fc recombinant protein was coated into an enzyme-labeled plate (working volume 100 ⁇ l) at 1 ⁇ g/ml, and left to stand overnight at 4°C. Wash 3 times with PBS (PBST) containing 0.05% Tween 20. Block with 5% skim milk in PBS for 1 hour at room temperature, and wash 3 times with PBST.
- the humanized antibody protein of h6H11B10#40 was diluted 1:5 with hIgG4 protein as a negative control and anti-LAG3 antibody BMS986016 as a positive control.
- LSECtin (R&D company) was prepared with 1 ⁇ g/ml, mixed with antibody at a ratio of 1:1, and allowed to stand at room temperature for 30min. Then add it to the microtiter plate and let stand at room temperature for 1 hour. Wash 3 times with PBST, add 100 ⁇ l horseradish peroxidase labeled mouse anti-His tag secondary antibody (GenScript) diluted 1:5000, and let stand at room temperature for 1 hour. Wash 6 times with PBST, add TMB for color development, and terminate the reaction with 2M H 2 SO 4. Use a microplate reader to read at 450nm. The results are shown in Figure 9, the anti-LAG3 humanized antibody h6H11B10#40 has the ability to block the binding of LAG3-Fc to its ligand LSECtin.
- Example 7 The ability of anti-LAG3 humanized antibody to block the binding of LAG3 to its ligand
- a commercial kit for determining the binding of LAG3 to FGL1 by anti-LAG3 antibodies (purchased from Cisbio, catalog number: 63ADK000CB10PEG) was used to test the ability of anti-LAG3 humanized antibodies to block the binding of LAG3 and FGL1.
- This experiment first mixes 4 ⁇ L of Tag1-LAG3 protein and 4 ⁇ L of Tag2-FGL1 protein with the diluted test sample, reacts at room temperature for 15 minutes, then adds 10 ⁇ L of pre-mixed anti-Tag1-Tb3+ and anti-Tag2-XL665, and seals the test. After reacting overnight at room temperature, read the fluorescence absorbance value of 665/620nm with a microplate reader. The results are shown in Figure 10, the anti-LAG3 humanized antibody h6H11B10#40 has the ability to block the binding of LAG3 to its ligand FGL1.
- the recombinant mouse and cynomolgus LAG3-Fc fusion protein was purchased from Yiqiao Shenzhou.
- the LAG3-Fc recombinant protein was coated into an enzyme-labeled plate (working volume 30 ⁇ l) at 1 ⁇ g/ml, and allowed to stand overnight at 4°C. Wash 3 times with PBS (PBST) containing 0.05% Tween20. Block with 5% skim milk in PBS for 1 hour at room temperature. Wash 3 times with PBST, add gradiently diluted anti-LAG3 humanized antibodies h6H11B10 and h6H11B10#40, and let stand at room temperature for 1 hour. At the same time, BMS986016 was used as a positive control.
- Flow cytometry was used to measure the binding of humanized antibodies to the cynomolgus monkey LAG3 on the cell surface, and CHO-S cells transfected and expressing the cynomolgus monkey LAG3 full-length gene were taken, and the cells were washed with PBS (2% FBS) Twice, adjust the cell concentration to 5 ⁇ 10 5 /100 ⁇ l. Add 1:3 gradient dilution of anti-LAG3 humanized antibody, and incubate at 4°C for 30min. At the same time, BMS986016 was used as a positive control. Centrifuge the cells at 500 ⁇ g for 5 min. Wash twice with PBS (2% FBS).
- SPR Surface plasmon resonance
- Example 9 The ability of anti-LAG3 humanized antibodies to activate T cells
- Example 10 Inhibitory activity of anti-LAG3 humanized antibody on tumor growth of hLAG3 KI mouse model
- hLAG3 KI mice Using hLAG3 KI mice, the ability of humanized anti-LAG3 antibodies to inhibit tumor cell growth in vivo was tested.
- Mouse colon cancer cells M38 were inoculated into the back of hLAG3 KI mice subcutaneously at 1 ⁇ 10 6 cells per mouse.
- the antibody has two doses of 30mg/kg and 10mg/kg, injected twice a week, about 2 to 3 weeks.
- the test groups include: isocratically diluted h6H11B10#40 sample group (the highest test concentration is 10 ⁇ g/mL) , Isocratic dilution HLX10 group (the highest test concentration is 10 ⁇ g/mL), in which the combination group is diluted with different concentrations of HLX10 solution (1 ⁇ g/mL, 0.1 ⁇ g/mL, 0.01 ⁇ g/mL and 0.001 ⁇ g/mL) h6H11B10# 40.
- the amount of IL-2 and IFN- ⁇ in the group treated with h6H11B10#40 or HLX10 alone is less.
- concentration of HLX10 reaches about 1 ⁇ g/mL
- IL-2 The release amount of and IFN- ⁇ is higher than that of h6H11B10#40 single-drug and HLX10 single-drug group, combined drug group (when HLX10 concentration is 1 ⁇ g/mL and 0.1 ⁇ g/mL), h6H11B10#40 shows a concentration-dependent effect.
- This in vitro experiment shows that the combination of h6H11B10#40 and HLX10 can reactivate exhausted T cells and increase the release of cytokines.
- CV coefficient of variation
- the TGI% is 62.2% and 56.7%, which are higher than the single-drug group.
- the A20 cells were cultured in the medium RPMI-1640+10% FBS, and the A20 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration (0.1 mL/mouse) for subcutaneous inoculation of mice.
- the experimental mice were subcutaneously inoculated with A20 cells (5 ⁇ 10 5 /mouse) on the right anterior scapula.
- the day of grouping was defined as day 0, and the administration started on day 0.
- the tumor volume was greater than 2500mm 3 and euthanized, resulting in a decrease in the number of mice.
- the 14th day was used as a TGI analysis.
- the TGI of h6H11B10#40 per kg and 30mg/kg h6H11B10#40 were 13.36%, 12.83% and 22.20%, respectively.
- the TGI% are 25.9% and 62.4%, respectively, which are better than the single-drug group, showing the combination group’s Anti-tumor effect.
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Abstract
Description
样品 | ka(1/Ms) | kd(1/s) | KD(M) |
BMS986016 | 3.22E+04 | 2.04E-04 | 6.35E-09 |
h6H11B10#40 | 4.35E+04 | 1.37E-05 | 3.13E-10 |
样品 | ka(1/Ms) | kd(1/s) | KD(M) |
h6H11B10#40 | 2.17E+04 | 3.09E-05 | 1.422E-09 |
Claims (15)
- 一种抗LAG3单克隆抗体,其特征在于,所述抗LAG3单克隆抗体包含重链可变区和轻链可变区,所述重链可变区包含与SEQ ID NO:2、6或10所示重链可变区具有的CDR序列相同的HCDR1、HCDR2和HCDR3区,和所述轻链可变区包含与SEQ ID NO:4、8或12所示轻链可变区具有的CDR序列相同的LCDR1、LCDR2和LCDR3。
- 如权利要求1所述的抗LAG3单克隆抗体,其特征在于,所述抗LAG3单克隆抗体包括:1)重链互补决定区HCDR1、HCDR2和HCDR3,所述HCDR1具有如SEQ ID No:18所示的氨基酸序列,所述HCDR2具有如SEQ ID No:19所示的氨基酸序列,所述HCDR3具有如SEQ ID No:20或21所示的氨基酸序列;2)轻链互补决定区LCDR1、LCDR2、LCDR3,所述LCDR1具有如SEQ ID No:22或23所示的氨基酸序列,所述LCDR2具有如SEQ ID No:24所示的氨基酸序列,所述LCDR3具有如SEQ ID No:25所示的氨基酸序列。
- 如权利要求2所述的抗LAG3单克隆抗体,其特征在于,所述抗LAG3单克隆抗体包括重链可变区和轻链可变区,所述重链可变区具有如SEQ ID NO:2、6或10所示的氨基酸序列,或者与上述序列具有至少85%同源性的序列;所述轻链可变区具有如SEQ ID NO:4、8或12所示的氨基酸序列,或者与上述序列具有至少85%同源性的序列。
- 如权利要求2所述的抗LAG3单克隆抗体,其特征在于,所述抗LAG3单克隆抗体包括重链和轻链,所述重链由具有如SEQ ID NO:2、6或10所示的氨基酸序列或者与上述序列具有至少85%同源性的序列的重链可变区和如SEQ ID NO:14所示的重链恒定区组成;所述轻链由具有如SEQ ID NO:4、8或12所示的氨基酸序列或者与上述序列具有至少85%同源性的序列的轻链可变区和如SEQ ID NO:16所示的轻链恒定区组成。
- 如权利要求1所述的抗LAG3单克隆抗体,其特征在于,所述抗LAG3单克隆抗体是抗体的全长序列或包含抗LAG3抗体的抗原结合片段,所述抗LAG3抗体的抗原结合片段为Fab、Fab’、F(ab’)2、Fv或scFv。
- 一种核苷酸分子,其特征在于,所述核苷酸分子编码如权利要求1至5中任一项所述的抗LAG3单克隆抗体。
- 如权利要求6所述的核苷酸分子,其特征在于,所述核苷酸分子中,编码抗LAG3单克隆抗体的重链可变区的核苷酸序列如SEQ ID NO:3、7或11所示,编码抗LAG3单克隆抗体的轻链可变区的核苷酸序列如SEQ ID NO:5、9或13所示。
- 一种表达载体,其特征在于,所述表达载体含有权利要求6或7所述的 核苷酸分子。
- 如权利要求8所述的表达载体,其特征在于,所述表达载体选自pHLX101、pEE14.4、pCHO 1.0或pcDNA3.1中的一种或多种。
- 一种宿主细胞,其特征在于,所述宿主细胞含有权利要求8或9所述的表达载体。
- 如权利要求10所述的宿主细胞,其特征在于,所述宿主细胞选自COS、CHO、HeLa细胞系、骨髓细胞系如SP2/0细胞系、NS0、sf9、sf21、DH5α、BL21(DE3)或E.coli TG1、YB2/0细胞系以及转化的B-细胞或杂交瘤细胞中的一种或多种。
- 一种制备权利要求1至5任一项所述的抗LAG3单克隆抗体的方法,包括以下步骤:a)在表达条件下,培养权利要求10或11所述的宿主细胞,表达抗LAG3单克隆抗体;b)分离并纯化步骤a)所得的抗LAG3单克隆抗体。
- 一种组合物,其特征在于,所述组合物包含如权利要求1至5任一项所述的抗LAG3单克隆抗体和药学上可接受的载体。
- 如权利要求1至5任一项所述的抗LAG3单克隆抗体或权利要求13所述的组合物在制备LAG3分子阻滞药物,尤其是制备抗肿瘤、治疗自身免疫性疾病、治疗感染性疾病和/或抗移植排斥反应的药物中的应用。
- 如权利要求14所述的用途,其特征在于,将所述抗LAG3单克隆抗体单独使用或与其他抗肿瘤药物联合使用,所述其他抗肿瘤药物选自抗体类药物,如抗PD-1单克隆抗体,或小分子抗肿瘤药物,如紫杉醇、5-Fu嘧啶。
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Application Number | Priority Date | Filing Date | Title |
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KR1020227028342A KR20220131279A (ko) | 2020-01-21 | 2021-01-19 | 항lag3단일 클론 항체 및 그 제조 방법과 응용 |
BR112022013544A BR112022013544A2 (pt) | 2020-01-21 | 2021-01-19 | Anticorpo monoclonal anti-lag3 e método de preparação do mesmo e uso do mesmo |
EP21744582.4A EP4095159A4 (en) | 2020-01-21 | 2021-01-19 | MONOCLONAL ANTI-LAG3 ANTIBODY AND METHOD FOR THE PRODUCTION THEREOF AND ITS USE |
US17/759,180 US20230074657A1 (en) | 2020-01-21 | 2021-01-19 | Anti-lag3 monoclonal antibody, and preparation method therefor and use thereof |
CA3165211A CA3165211A1 (en) | 2020-01-21 | 2021-01-19 | Anti-lag3 monoclonal antibody, and preparation method therefor and use thereof |
CN202180008891.3A CN114945595A (zh) | 2020-01-21 | 2021-01-19 | 抗lag3单克隆抗体及其制备方法和应用 |
JP2022544146A JP2023510982A (ja) | 2020-01-21 | 2021-01-19 | 抗lag3モノクローナル抗体、その製造方法および使用 |
AU2021210029A AU2021210029A1 (en) | 2020-01-21 | 2021-01-19 | Anti-LAG3 monoclonal antibody, and preparation method therefor and use thereof |
ZA2022/08339A ZA202208339B (en) | 2020-01-21 | 2022-07-26 | Anti-lag3 monoclonal antibody, and preparation method therefor and use thereof |
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EP (1) | EP4095159A4 (zh) |
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KR (1) | KR20220131279A (zh) |
CN (1) | CN114945595A (zh) |
AU (1) | AU2021210029A1 (zh) |
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WO2023138677A1 (en) * | 2022-01-24 | 2023-07-27 | Crown Bioscience Inc. (Taicang) | Novel anti-lag3 antibodies and derivative products |
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CN114945595A (zh) | 2022-08-26 |
EP4095159A4 (en) | 2024-03-20 |
BR112022013544A2 (pt) | 2022-09-06 |
ZA202208339B (en) | 2023-11-29 |
EP4095159A1 (en) | 2022-11-30 |
JP2023510982A (ja) | 2023-03-15 |
AU2021210029A1 (en) | 2022-09-01 |
KR20220131279A (ko) | 2022-09-27 |
CA3165211A1 (en) | 2021-07-29 |
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